Over the past 30 years, development of novel diagnostic apparatuses and methods which involve quantitative and qualitative analyses of extremely small quantities of substances contained in a sample taken for biopsy, such as blood or urine, has actively and rapidly progressed and even now, is still progressing at a high speed. RIA (Radioimmunological Assay) using radioactive isotopes was introduced in the 1950s, and ELISA (Enzyme Linked ImmunoSorbent Assay) was developed and advanced in the 1970s and 1980s. The ELISA method is the most popular laboratory test today and one of requisite tools for research in medical or life science fields. Recently, modified ELISA methods have been developed. Among them, for example, there is a method for analyzing a plurality of analytes at one time by immobilizing a plurality of antibodies onto a 96-well plate.
By typical immunodiagnostic methods, including RIA or ELISA, only one kind of analyte per sample can be quantified, using expensive analytical machinery and tools, while performing a multi-step procedure. Therefore, these methods cannot be readily used in a small-scale hospital, emergency room, the home, etc., where such equipments are not provided. In order to make up for this weak point, a convenient diagnostic kit using immunochromatography has been developed.
Using such diagnostic kit, it is possible to obtain a test result in 15 minutes after applying a sample such as whole blood, serum, urine, etc. to the kit. A representative type of immunochromatographic assays is a lateral flow assay. A kit for the lateral flow assay has a structure comprising a sample pad, to which a sample is applied, a releasing pad coated with a detector antibody, a developing membrane (typically, nitrocellulose) or strip, in which components of the sample move at different rates to be individually separated and to undergo antibody-antigen reaction, and an absorption pad which is provided at the far end of the sample pad to cause the sample to keep moving. The detector antibody is fixed onto, for example, colloidal gold particles to enable the detection. Latex beads or carbon particles may be used instead of gold particles. The diagnostic kit for the lateral flow assay is generally designed to detect an analyte in a sandwich configuration. Upon applying a liquid sample to the sample pad of the kit, an analyte contained in the sample begins to move from a sample pad. Firstly, the analyte reacts with a detector antibody releasably adhered to a releasing pad to form an antigen-antibody conjugate, which continues to develop in this conjugated form. Then, while moving through the developing membrane, the antigen-antibody conjugate reacts once more with a capture antibody fixed on a developing membrane to form a capture antibody-antigen-detector antibody conjugate in a sandwich form. Since the capture antibody is fixed on the developing membrane, conjugates are accumulated in the area where the capture antibodies are fixed. Proteins are invisible to the naked eye. Therefore, the presence and amount of conjugates are determined by means of an amount of gold or silver particles attached to a certain area of the developing membrane.
The lateral flow assay can be widely and conveniently used in various fields such as pregnancy diagnosis, cancer diagnosis, and microbe detection. However, since quantification cannot be performed with the naked eye and hence, an exact amount of an analyte cannot be determined, its application is restricted. Especially, when a judgment should be made around a cut-off value, it is difficult to make an exact diagnosis. For example, in case of prostate cancer, when a detected value is 3.9 ng/ml which is very close to the standard cut-off value of 4 ng/ml, an exact diagnosis cannot be made.
Immunodiagnosis is now rapidly developing, and in the near future, will be able to easily and promptly identify and analyze a sample. Therefore, we can diagnose the condition of disease. The RIA or ELISA method which can quantify an analyte at present involves several complicated steps for such quantification, including treatment with an enzyme and washing. Similarly, the conventional convenient diagnostic kits have difficulties in providing quantified results. Therefore, there is a great demand for a general assay method which can perform quantification more rapidly, conveniently and sensitively. With the method, an ordinary unskilled person can practice diagnosis or analysis in any place.
In addition, the immunoassay technology is an attractive method for qualitatively and quantitatively detecting target substances contained in biological samples using DNA chips or protein chips incorporating membranes in a short time and at low cost. Since diagnostic chips produced for such analysis selectively detect biomedical markers generated when a specific disease develops, they are very important in diagnosing a target disease and can provide information on abnormal conditions. Diagnostic chips used in such an immunoassay technology greatly simplify the conventional pathological tests with respect to time, space and procedure. However, most chips including the conventional lateral flow quantitative assay strips still allow only qualitative analysis. That is, with these chips, immunological reaction results are converted to visually identifiable forms and interpreted based on the subjective criteria of analyzers. At present, this assay is gaining popularity due to its convenience of not requiring specific analysis apparatuses. A representative example is a pregnancy diagnostic chip.
Diagnostics should be performed by precise qualitative or quantitative assays. At present, available representative analysis tools are spectrometry and fluorescence analysis, which are applied for high-throughput screening. A representative example of the fluorescence analysis is laser-induced fluorescence detection. The laser-induced fluorescence detection technique is based on exciting a fluorescent material from a ground state to an excited state using a laser light of a wavelength absorbed by the fluorescent material, and measuring the intensity of fluorescence emitted upon the return of the electronic energy state from the excited state to the ground state, whereby the measured fluorescence intensity indicates the concentration of the fluorescent material. In this way, DNA and protein samples that are tagged with fluorescent materials are quantitatively analyzable.
On the other hand, DNA chips contain DNA molecules with various lengths ranging from several hundreds to hundreds of thousands of base pairs in a very small space by means of a mechanical automation or electronic control, etc. That is, DNA chips are biological microchips that are capable of analyzing gene expression patterns, gene defects, protein distribution, response patterns, and the like, using DNA molecules attached to a small support made of a transparent or semi-transparent substance, such as a glass or silicon. DNA chips are classified to two categories according to the size of the genetic material attached thereonto: cDNA chips and oligonucleotide chips. A cDNA chip contains at least 500-bp or longer full-length open reading frames attached thereonto, and an oligonucleotide chip contains oligonucleotides consisting of about 15 to 25 bases.
In general, there are two types of DNA chip manufacturing technologies using target DNA molecules: direct synthesis of oligonucleotides on a support and immobilization of amplified target DNA molecules onto a support. The on-chip synthesis of DNA molecules, which is based on a photolithographic fabrication technique employed in the semiconductor chip industry, allows high density deposition of the DNA molecules, but the target DNA molecules are limited to be about 20 nucleotides in length. The photolithographic-based DNA chip manufacturing technology is suitable for disease diagnosis or single nucleotide polymorphism (SNP). The second technology is commonly applied to differential gene expression studies, and immobilizes target DNA molecules onto a slide coated with poly L-lysine, amine or aldehyde.
Protein chip fabrication techniques and applications are known to those skilled in the art and also described in many journals and patent publications. For example, a protein chip is manufactured by depositing antibodies to proteins associated with several diseases onto a small transparent or semi-transparent wafer, and can be used for early diagnosis for the presence and pathogenic states of specific diseases by being treated with an analyte, prepared using a body fluid collected from a patient, and applied to the chip as a biochemical marker. The small wafer is prepared by immobilizing a desired protein onto a common glass plate, for example, using avidin. Also, the wafer is prepared using polystyrene as a wafer substrate, and this polystyrene binds proteins with high efficiency. Polyvinylchloride and polypropylene are also used according to the nature of proteins immobilized onto a wafer.
A process of depositing proteins onto the aforementioned transparent or semi-transparent wafer is well known to those skilled in the art. For example, in case of using a polystyrene wafer, eight grooves 1 mm wide, 2 mm long and 1.5 mm deep are created in a row at intervals of 1 mm on a polystyrene wafer 1.5 cm wide and 1.5 cm long. When proteins to be analyzed are individually deposited in the grooves of the polystyrene wafer with a diameter of about 400 nm and intervals of about 500 nm, ten proteins can be deposited in a 1-cm length of the wafer. In total, about 80 proteins can be deposited onto the single wafer.
On the other hand, a confocal laser scanning system is most commonly used in detecting fluorescence using the laser-induced fluorescence detection technique. With this system using a laser as a light source, only fluorescence emerged from a single position of a specimen, among fluorescence signals emitted from the specimen, enters a photomultiplier tube by a specific photometric system, and the output from the photomultiplier tube, which is an analog electrical signal, is converted to a digital image.
That is, as shown in FIG. 10, the confocal laser scanning system uses a laser light source 11 to illuminate only a light with a proper wavelength a specimen labeled with a fluorescent material and emit fluorescence in the specimen. This system is designed to detect only fluorescence emitted from a fluorescence point by finally passing fluorescence emitted from an area several tens of micrometers containing the specimen through a pinhole 16 formed in front of an optical detector 17. The reference numerals 12, 14 and 15 indicate a spatial filter for incident light, an objective lens and the specimen, respectively.
Most fluorescence scanners employing this principle provide information on the intensity and spatial distribution of fluorescence emitted in a diagnostic chip with high precision, but have difficulties in use for point of care (POC) diagnostics, as follows. First, these kinds of scanners are supplied with a high price of 50,000 dollars, thereby making it difficult for the convenient diagnostic chips to be widely used. Also, the fluorescence scanners are not convenient products in use because they must be maintained under a stringently managed environment to execute the high sensitivity detection. These problems with the desk-top scanners can be partially overcome by employing a small-sized scanner. A small scanner called “Triage” from Biosite Diagnostics Inc. is available along with a diagnostic chip, which provides rapid quantitative measurements for cardiac markers, but is cost-ineffective because of providing high-priced diagnostics, for example, at 50,000 won per test in Korea. Response Biomedical's RAMP diagnostic system is the closest technology to the laser-induced fluorescence detection technique and fluorescence immunological cancer diagnostic devices using the technique, but is to date not commercialized.